Solid-phase synthesis support

ABSTRACT

A SUPPORT MEDIUM CONSISTING OF A HIGHLY SWELLABLE POLTMER RESIN COATED ON A FLUID IMPERVIOUS, SHAPED CORE MATERIAL IS DESCRIBED, IN SPEECIFIC EMBODIEMENTS. GLASS BEADS ARE PROVIDED WITH A COATING OF A HIGLY SWELLABLE LOWCORSS LINKED POLYMER SUCH AS POLYSTYRENE. REACTIVE GROUPS, SUCH AS THE CHLORIOMETHYL GROUP, ARE INTRODUCED INTO THE POLYMER COATING. THE AFORESAID SUPPORT MEDIUM IS USEFUL IN THE SOLID PHASE SYNTHESIS OF COMPLEX MOLECULES SUCH AS POLYPEPTIDES, POLYNUCLEOTIDES, POLYSACCHARIDES, PROTEINS, POLYAMIDES AND THE LIKE.

United States Patent Oifice 3,817,777 Patented June 18, 1974 3,817,777SOLID-PHASE SYNTHESIS SUPPORT Csaha Horvath, Orange, and Seymour R.Lipsky, Woodbridge, Conn., assignors to Hoifmann-La Roche Inc., Nutley,NJ.

No Drawing. Application Dec. 12, 1970, Ser. No. 100,368,

now Patent No. 3,725,111, which is a continuation-inpart of abandonedapplication Ser. No. 14,182, Feb. 25, 1970. Divided and this applicationAug. 30, 1972, Ser.

Int. Cl. C030 17/32 US. Cl. 117-100 S 7 Claims ABSTRACT OF THEDISCLOSURE RELATED APPLICATIONS This is a division of application Ser.No. 100,368 filed Dec. 12, 1970, now US. Pat. 3,725,111, issued Apr. 3,1973, which in turn is a continuation-in-part of Ser. No. 14,182 filedFeb. 25, 1970, now abandoned.

BACKROUND OF THE INVENTION The concept of utilizing solid-phasesynthesis procedures for the preparation of biologically active peptidesand proteins was first advanced by Dr. R. B. Merrifield. He recognizedthe need for a rapid, quantitative, automatic method for the synthesisof long-chain peptides. He conceived of a procedure which involvesattaching the initial component amino acid to an insoluble polymericmaterial and then building up the chain to the desired length'by thesequential addition of various activated protected amino acids in apredetermined order. Each intermediate cycle would involve removal ofthe protecting group on the last amino acid on the chain followed bycoupling of the next selectively protected amino acid until the desiredpeptide chain is obtained. In a final step the peptide would be removedfrom the support medium. An excellent summary of this procedure iscontained in Dr. Merrifields Lasker award lecture published in theJournal of the American Medical Association, Vol. 210, No. 7 (Nov. 17,1969) at pages l247l254.

The disclosure of Dr. Merrifields work prompted an extensive amount ofresearch by other workers with the aim of developing improved polymersystems to act as support in the solid-phase synthesis. Work was alsodirected to improving the amino acid blocking groups, the reactionconditions employed in adding and splitting olf of protecting groups,the conditions used in linking the initial amino acid to the polymer andthe conditions for the ultimate removal of the complex molecule from thepolymer chain. The extensive nature of this work is well summarized inDr. Merrifields review article entitled Solid-Phase Peptide Synthesis,Advan. Enzymol. 32: 221-296, 1969.

The advantages which are potentially available from the utilization of asolid-phase synthesis system include the simplification and accelerationof multi-step syntheses because of the possibility of being able tocarry out all the reactions in a single reaction vessel thereby avoidingthe manipulations and attendant losses involved in repeated transfer ofmaterials; the avoiding of large losses normally encountered duringisolation and purification of intermediates since such intermediates arebound to an isoluble phase throughout the synthesis; acceleration of thepurification of intermediate products because they do not have to beisolated; the amenability of such systems to adaptation of automated andmechanized procedures; the obtaining of high yields of final product ofgood purity through the use of excess reactants to force the individualreactions to completion; and the ability to prepare compoundssynthetically which otherwise would not be readil obtainable byconventional methods.

However, in order to bring such advantages to practical fruition it isnecessary that the polymeric support utilized meet several stringentrequirements. For example, it is necessary that such support materialsbe completely insoluble in all solvents which are used in the synthesisand should be both chemically and physically stable. Additionally, thepolymeric material must possess a structure and form which permits rapiddifusion of reagents into the reactive site and easy removal of reagentsand byproducts by filtration and washing. The support must have largepore dimensions in order to facilitate completion of each step of thesynthesis even if the growing peptide chain contains a large number ofamino acids. Finally, the support must contain a functional group whichwill allow ready attachment of the first synthetic component, e.g., thefirst amino acid b a stable, covalent bond.

The solid supports most generally employed in solidphase synthesis arecurrently based on a copolymer of styrene and divinyl benzene. Thesematerials are gels with a random network of loosely cross-linkedpolystyrene chains. Modifications in this system have generally beendirected to the degree of cross-linking, physical form, or addedfunctional groups. It is to be noted that the degree of cross-linkingdetermines the extent of swelling, thus the eifective pore size, and themechanical stability of the support. These properties in turn determinedthe suitability of the polymers for solid-phase synthesis. It has beenobserved in the art that polymers containing from 1 to 2% cross-linkingare generally most preferable since reducing the amount of cross-linkingbelow this level has resulted in polymer particles of poor mechanicalstability which fragment during the synthesis and of which purificationand filtration proved to be extremely difiicult.

In order to overcome diffusion limitations restricting free access ofreactant molecules to reactive sites on the polymer support which areexhibited to a substantial degree with the l to 2% cross-linkedpolymers, the prior art has tried macroporous, also calledmacroreticular, resins. These resins are rigid copolymers containinglarge pores fixed by a high degree of cross-linking. However, theseresins proved to be inadequate for the synthesis of complex large chaincompounds in that a progressively decreasing extent of reaction wasobserved as the peptide chain was lengthened. It was concluded that agel-like polymer that swells freely would provide a less hindered matrixand therefore a more satisfactory support.

A further possibility that was explored was to utilize low cross-linkedpolymers that were in a popcorn rather than a bead form. It was found,however, that while the initial velocity with the popcorn polymer wasfaster than for the beads, the reaction did not go to completion and itwas apparent that a steric barrier was present. Additionally, thepopcorn polymer was found not to swell appreciably in organic solventsand were of low density thereby making their use with high densitysolvents less desirable. This work is summarized in several papers byLetsinger and various co-workers appearing in J. Polymer Sci. 11-], 5,1977 (1967); J. Am. Chem. Soc. 85, 3045 (1963); and J. Am. Chem. Soc.86, 5163 (1964).

3 BRIEF DESCRIPTION OF THE INVENTION The present invention relates to animproved solid support medium, its use in the solid-phase synthesis ofcomplex molecules and to methods for its preparation. In particular, thesupport medium of the present invention comprises a swellable, lowcross-linked polymer coated onto a mechanically stable, dense, fluidimpervious shaped support. The polymer coating is characterized incontaining reactive functional groups which can couple with the firstcomponent molecule of the complex compound to be synthesized. Thesupport medium of the present invention exhibits properties whichprovide it with substantial advantages over the solid supportsheretofore known in the solid-phase synthesis art. The polymer coatingof the present solid support medium is cross-linked to a very low leveland thus exhibits substantial swelling leading to a more open polymerstructure. In turn such swelling characteristics result in superiorkinetics for the initial coupling reaction onto the polymer as well asfor the chain addition reactions used in the building up of the complexmolecule. This swelling ability is not obtained by sacrifice ofmechanical stability of the structure and thus the support materials ofthe present invention are especially amenable to use in column systemsthereby facilitating its adaptation to automated, continuous flowprocedures.

The dense, fluid impervious shaped support forming the core of thesupport medium of the present invention can be selected from a widevariety of materials. Suitable fluid impervious core materials includemetals, such as iron, aluminum, copper, silver, gold, lead and the like;metal alloys, such as steel, bronze, brass and the like; metal oxidessuch as aluminum oxide, silicates and the like; ceramic materials,clays, wood; highly cross-linked resins which may comprise similar ordissimilar polymers utilized in the outer coating of the supportmaterial; glass and like materials. The aforesaid core materials may beshaped in any form; particularly those which are conventionally employedin column packing. Suitable shapes include, for example, spheres,Raschig rings, Berl saddles, Steadman packings, wire mesh rings, wirehelices and the like. For most purposes in the practice of the presentinvention it is preferable to utilize spherically shaped fluidimpervious core materials. Glass beads represent a particularlypreferred embodiment of this aspect of the present invention.

The polymer coating of the support medium of the present invention cancomprise polymeric material conventionally used in solid-phase peptidesynthesis procedures known in the art. Examples of suitable polymersinclude polymers derived from styrene, phenol-S-trioxan, methylmethacrylate, vinyl alcohol, cellulose, dextran, vinyl toluene, vinylxylene, vinyl naphthalene, vinyl ethyl benzene, wmethyl styrene, vinylchlorobenzene, vinyl dichlorobenzene or mixtures thereof. The aforesaidpolymers are cross-linked utilizing conventional cross-linking agentssuch as divinyl benzenes, divinyl toluenes, divinyl xylenes, divinylnaphthalenes, divinyl ethyl benzenes, etc. In order to provide thedesirable swelling characteristics of the solid supports of the presentinvention, it is preferred that the aforesaid polymers be cross-linkedin the range from about 0.01 to 2.00 percent, most preferably in therange of from about 0.05 to 0.50 percent. For the purposes of thisapplication the term percent of cross linking is meant to mean the molepercent of cross-linking agent added to the polymerization mixture. Ahighly preferred polymer useful in coating the solid support of thepresent invention is a polystyrene cross-linked with divinyl benzene.

The low cross-linked polymer can be applied to the dense fluidimpervious core material by a variety of methods. For example, a polymerresin in the form of beads can be coated onto the desired core materialby utilizing the procedure of impact coating such as described in US.Pat. No. 2,788,297 issued Apr. 9, 1957 to Louis. Alternatively, thepolymer can be applied in the form of a suspension or a paste to thecore material whose surface, if desired, can be pretreated with alacquer or varnish. This technique is described in some detail in US.Pat. No. 3,340,085 issued Sept. 5, 1967 to Halasz et al. In anothertechnique the dense, liquid impervious core is a cross-linked vinylpolymeric material such as polystyrene prepared with from about 10 to30% of a polyvinyl cross-linking agent such as divinyl benzene. Thesurface of this core material is then coated with the same or adifferent polymeric material which is then crosslinked with across-linking agent in the range of from about 0.01 to 1.00 percentthereby yielding the desired swellable, low cross-linked surface. Thisprocedure is analogous to that described in US. Pat. No. 3,252,921issued May 24, 1966 to Hansen et al. In a further, most preferredtechnique, a polymer matrix is rendered onto a suitably treated surfaceof the dense, fluid impervious core material in the form of a thin-layerby polymerizing the monomer mixture of the glass bead surface in situusing a technique resembling a suspension (pearl) polymerization method.This latter procedure is discussed in further detail at a further pointin this specification.

It is generally desirable that the low cross-linked polymer coated solidsupport medium of the present invention contain from about to 99.8weight percent of the dense, fluid impervious core, most preferably fromabout to 99.8 weight percent, and from about 0.2 to 20 weight percent ofthe low cross-linked polymer coating, most preferably from about .02 to5 weight percent.

In order to be able to couple the first component of the complexmolecule being synthesized to the polymer it is necessary to providesuitable reactive groups on the polymer surface. A particularlypreferred reactive group for use in the present invention is thechloromethyl group which can be introduced by chloromethylating thepolymer with chloromethyl methyl ether and a Freidel-Craft catalyst,e.g., stannic chloride. Other reactive groups which may be employed inthe practice of the present invention include the following:

ll noornomomo-Qn, owm Q-R cums GHQ-R, ClSOz-Q-R II 010 0 GET-QR, o1-hnand the like.

In a preferred embodiment of the present invention low cross-linkedpolystyrene coated glass beads are employed in the solid-phase sythesisof complex molecules. The polystyrene polymer is chloromethylated inthis preferred embodiment to supply the necessary functional groups tobe used in coupling with the initial component of the complex moleculeto be synthesized. The preparation of such polystyrene coated glassbeads can be readily accomplished by a multistep process wherein thepolymer matrix is formed in situ by polymerization of the monomermixture on the glass bead surface. The steps in this process may beoutlined as follows:

(a) Purification of glass beads (b) Surface treatment of glass beads (c)Polymerization of monomer mixture on the surface of glass beads (d)Purification of the polymer (e) Swelling of the polymer (f)chloromethylation (g) Purification of the chloromethylated resin.

The purification of the glass beads generally includes an initialremoval of any iron particles by use of magnets followed by washing ofthe glass beads with a detergent solution and water. The beads are thenrinsed with a water miscible organic solvent such as a lower alkanol,most preferably methanol and then dried.

The surface treatment of the glass beads involves an attachment ofpreferably organosilicon compounds to the glass surface by chemicalreaction or adsorption in order to promote adhesion of the polymer tothe glass. It is conveniently accomplished by stirring the dry beads ina dilute solution, e.g., of a vinyl trihalo silane such asvinyltrichlorosilane in an aromatic hydrocarbon, e.g., benzene, atreflux for one to two hours. The beads are then filtered and rinsed witha lower alkanol such as propyl alcohol and dried.

The resin layer to be rendered onto the glass beads comprisespolystyrene cross-linked at from 0.01 to 2.00 percent, most preferablyfrom about 0.05 to 0.50 percent with a cross-linking agent. Any of theconventional crosslinking agents are suitable for this purpose, withdivinyl benzene being the cross-linking agent of preference. For thepurposes of this invention it is possible to utilize technical gradedivinyl benzene which contains various divinyl benzene isomers and otherbenzene compounds as impurities. Generally a content of about 50%divinyl benzene isomers will be adequate for this purpose. The degree ofcross-linking is defined as the weight percent of the cross-linkingagent, based on the amount of styrene, added to the monomer mixture. Itis to be noted that the amount of cross-linking agent which is presentin the polymer prepared by the present method may be more, equal or lessthan the fraction originally put into the monomer mixture. Whenutilizing technical grades of cross-linking agent, it is understood, ofcourse, that only the fraction of the pure divinyl benzene or othercross-linking agent contained in the monomer mixture is to be used tocalculate the degree of cross-linking.

The polymerization reaction may be initiated by conventional initiatingagents. Suitable initiating agents for this purpose include the organicperoxides, such as benzoyl peroxide and ditertiary butyl peroxide.Typical monomer mixtures which can be utilized in the preparation of thepolymer coated glass beads for the present invention are given in thefollowing recipe:

1000 to 2000 parts of distilled styrene;

1 to 30 parts of technical divinyl benzene containing 50% divinylbenzene isomers;

3 to 100 parts of peroxide initiator.

Thus, for example, a suitable monomer mixture may contain 1,000 parts ofstyrene, 5 parts of technical divinyl benzene and parts of benzoylperoxide to yield a 0.25% cross-linked polymer coating.

The polymerization process is most desirably carried out in a mannerthat will serve to achieve satisfactory bonding between the treatedglass surface and the polymer. It is further desired that the productconsist of individually separated glass beads each covered with therequisite polymer coating. It is therefore required that thepolymerization process, or at least the final stage 6. thereof, when thepolymer becomes hard, be carried out under conditions where the beads,which are covered with the monomer mixture or with a lightly polymerizedmixture, are separated from each other. This is achieved by carrying outthe entire polymerization or at least its final stage under vigorousstirring in suspension in an aqueous medium containing a suspendingagent.

A particularly preferred mode of effecting the polymerization procedureinvolves utilizing from 50 to 500, most preferably from to 200, parts ofglass beads pre-treated as above mixed with from about 2 to 100, mostpreferably from about 5 to 30, parts of the desired monomer mixture. Themixing should be so intensive that the surface of the heads is wettedcompletely with the monomer mixture. The wet glass bead massthusobtained is placed into a resin kettle equipped with a refluxcondenser, thermometer and a strong stirrer. Then from to 3000, mostpreferably from 250 to 1000, parts of an aqueous solution containingfrom about 148 to 2900, most preferably from about 245 to 920, parts ofwater and from about 2 to 100, most preferably from about 5 to 80, partsof a suspending agent such as a mixture of polyvinylpyrrolidone orpolyvinyl alcohol with a surfactant such as sodium lauryl sulfate isadded.

The resin kettle is heated with a water bath to about 70 C. and theglass beads wetted with the water and soluble monomer are stirredoccasionally. When the gelation of the monomer begins, which isrecognized by an increase in the monomer viscosity and occurs within twohours from the beginning of the heating, the stirrer is turned on andfrom then on the content of the kettle is stirred vigorously andcontinuously.

After about three and one-half hours of stirring, the reaction mixtureis cooled to room temperature. The supernatant milky dispersion of thepolymer which has been washed off from the beads during the process isseparated and the product is washed several times with water so that thefinally dispersed polymer particles and suspending agent attached to thesurface of the beads are completely removed. The product is dried in airor in a vacuum drying oven at a temperature not higher than 80 C. Theproduct consists of polymer coated glass beads, of aggregates of suchbeads and resin particles which have been detached from the beads duringhandling of the product.

In a particularly preferred embodiment 10 parts of glass beads, 1 partof the styrene monomer mixture and 25 parts of an aqueous solutioncontaining 22.5 parts of water and 1.5 parts of suspending agent areutilized. This recipe yields about 10 parts of intermediate product.

If desired, the polymerization procedure can be repeated subsequentlyusing polymer coated beads as starting material in order to increase thethickness of the polymer layer.

The product obtained above may be sieved to remove aggregates and fines,although the material can also be used without screening in furtherreaction steps. Generally, it is desired to utilize a screen having amesh in the range of that used for screening the starting glass beads.After screening, the yield based on about 10 parts of intermediateproduct is about 8 to 9.5 parts.

The product can be purified by washing or by treatment in a Soxhletextractor with a solvent which swells the polymer well such as anaromatic hydrocarbon such as benzene, toluene etc. In this treatment,the monomers, oligomers and the soluble polymer fractions which remainin the resin are removed.

Prior to effecting chloromethylation of the polymer coated glass beadsit is necessary that the beads first are swelled in chloromethyl methylether. The swelling can be accelerated by utilizing elevatedtemperatures, e.g.,

a temperature in the range of from about 30 to 60 C.

After the resin layer on the beads is adequately swollen, thechloromethylation is carried out with a mild Friedel- Craft catalyst,for example, stannic chloride. Although the introduction of a largenumber of chloromethyl groups is desirable to impart high capacity tothe resin for the solid state synthesis, the reaction should beterminated before intramolecular cross-linking occurs by the formationof methylene bridges. The chloromethylated product is The tertiarybutyloxycarbonyl group may then be removed by mild hydrolysis such as bytreating with hydrochloric acid-acetic acid using conditions known inthe art. The protective group is split olf under these conditions butthe initial bond of the amino group to the polymer is not purified inorder to remove the catalyst, soluble fractions ff cted, A second aminogroup may then be coupled to of the polymer, paraformaldehyde and otherby-products. the first amino group by utilizing an activating compoundGenerally the resulting Product Will contain from 2 to 25 such asdicyclohexylcarbodiimide and then the protecweight percent of chlorinebased on the weight content of tive group from the second amino acid maybe removed in polymer in the coated material. 10 similar manner to thatdescribed above. This process can 111 a sPeethe embodiment 1,000 of P yCoated be continued until the desired sequence of amino acids glassbeads are suspended y gentle stirring in 700 have been built up. In thefinal ste the peptide is re- Of distilled chloromethyl methyl etherplaced in a round moved from the polymer by treating with hydrobromic orbo tom fl and heated to boiling 0- Then hydrochloric acidtrifluooracetic acid. The resulting the vhatch is cooled to 1130111temPefetul'e and a solution peptide is then recovered from the solutionin relatively of 18 ml. of distilled stannic chloride in 50 ml. ofchloropure f methyl methyl ether is added drcpwise to the suspension Thesolid-phase synthesis of complex molecules utilizing The temperature israised slowly to the g Point and the support medium of the presentinvention can be conthe suspension is stirred under reflux for twohours- A t ducted in reaction vessels conventionally employed in thecooling, the beads are filtered onafritted glass filter funnel 20 artfor this purpose such as, for example, in shake or and Washed wi a totalof 1,000 of diOXalle in three rocker flasks. However, it is alsopossible to utilize the P Then the heads are pen e in 2,000 of presentsupport medium as a fixed bed in a column reactor 8. mixture co g of 90%dioxane and 10% concenwhich has heretofore not been possible with thelow cross trated hy r chl ri i The Product is filtered again linkedolymeric materials of the art due to their poor and washed on the filterwith 1,000 ml. of the above mixphysical stability properties and theresulting filtration ture in small portions. Afterward the beads arewashed difli 1ti with a total of 1,000 ml. of dioxane in three portionson A great advantage deriv bl fro th use f column the filter and theproduct is then air dried. technology in solid-phase synthesis is thefact that it is The solid support medium prepared in acco a ce possibleto employ a process wherein reactants and rewith the present inventionis generally useful in solidagents are passed through the column of thepresent supphase synthesis of complex molecules. In such synthesis t diuin onti o f hi n in th d sired s a poly-fllhetiohal first eompoheht ofthe complex molecule quence. Thus, the time required for the synthesisof a long is uti ize o Of e functional g p of this first chained productis substantially reduced. Moreover, introponent is provided in free formsuitable for coupling with duction of a fully automated control systemfor this the reactive group associated with the p y coating on procedureis more facile than for the alternate methods of the solid supportmedium while the remaining one Or more operation previously useable inthe art, functional groups of the first component are provided in Thepresent invention is further illustrated by the folprotected form. Thefirst step in the solid-phase synthesis lowing examples. procedureinvolves the coupling of the first component to EXAMPLE 1 the polymersurface via the free functional groups rep y available e the c p has e hachieved, This example demonstrates the superior swelling propthefunctional gfohp'htlhzed 111 further addlhohs to t erties ofrepresentative polymer coated glass beads of Component molecule is freedy removal the pl'htectlve the present invention when compared to acommercial- S P- Then the Second component Provided with e ly availablepolystyrene bead conventionally employed free functional S P andl'emainlhg groups hlhcked 15 in solid-phase synthesis by the art. Bead Aused in these added to the first eempoheht- The P f is then f testsrepresents the commercial polystyrene bead which Peated with the secondeempeheht Proteetlve g p helhg is chloromethylated to a chloride contentof 2.08 mm./g. removed and added components reacted sequentially ofpolymer and is cross-linked to a level of about 2% with thereto yrepetitithl 0f the above P a final Step divinylbenzene. Bead Brepresents a glass bead coated the completed complex molecule is removedfrom the with a polystyrene cross-linked to a level of 0.25% with P y Itshould he noted that one of the y features divinylbenzene,chloromethylated to a chloride content of a solid-Phase synthesisProcedure resides in the fact of 0.04 mm./g. of coated glass bead andwhich final t at t e b selected for p g the first component to headcomposition comprises about 99.10 weight percent the resin requires moresevere conditions for decoupling glass and 0.90 weight percent polymer.Finally, Bead than is required for the removal of the intermediate Crepresents a glass bead coated with polystyrene crossblocking groupsfrom the growing chain of components linked to a level of 0.25% withdivinylbenzene, chloroadded to the first component. methylated to achlorine content of 0.11 mm./g. of

Therefore, for example, a polypeptide may be synthecoated glass bead andwhich final bead composition comsized by first adding a protected aminoacid, e.g., an prises about 84.58 weight percent glass and 15.42 weightamino acid whose free amino group is protected with a percent polymer.tertiary butyloxycarbonyl group, to a chloromethylated Equal volumes ofthe aforesaid beads were swelled with polymer coated support medium ofthe present inven a variety of solvents and the swelling capacitydetermined. tion. The coupling reaction produces a protected amino Theresults of these tests are summarized below in group bonded by an acylgroup to the polymer chain. Table I.

TABLE I Bead A Bead B Bead C Total Free. vol Total Free. vol. TotalFrac. vol.- Dry Wet frac. inc. due to Dry Wet Irac. inc. due to Dry Wettrac. inc. due to vol., vol., vol. polyvol., vol., vol. polyvol., vol.,vol. polycc. cc. iner. styrene cc. cc. iner. styrene cc. cc. incr.styrene 10 25 2.5 2.5 10 12 1.2 133 10 as 3.3 21.4 10 25 2.5 as 10 121.2 133 10 a1 3.1 20.1 10 24 2.4 2.4 10 12 1.2 133 10 28 2.8 18.2 10 232.3 2.3 10 12 1.2 133 10 22 2.2 14.3

1 Total fractional volume increase/traction of polystyrene present:

Examination of the above results indicates that the fraction of volumeincrease to the polystyrene present in Bead B and Bead C indicatesvolume increases of 133 and 20 times respectively compared to a volumeincrease for Bead A of about 2.3 to 2.5 times. The increasedswellability of the polymer portion of the support medium of the presentinvention renders these materials extremely suitable to solid-phasesynthesis wherein difiusion based limitations are minimized.

EXAMPLE 2 This example demonstrates the efiicient manner in which theaddition of a first blocked amino acid compound cobonded to achloromethylene low cross-linked polystyrene coated glass bead of thepresent invention can be accomplished. The glass bead utilized in thisexample is Bead B described in Example 1.

In a 100 ml. three-necked flask fitted with a mechanical stirrer andreflux condenser with drying 'tube, were placed 172 mg. (0.792 mm.) ofBoc-valine, 20.0 g. of Bead B, 20 ml. of ethanol and 0.1 ml. (0.713 mm.)of triethylamine (added last). With the flask immersed in an oil bath at90 C., the mixture was stirred gently by means of a mechanical stirrerfor 60 hours. During the stirring process, the stirrer blade waspositioned above the surface of the beads to prevent shearing of thepolymer, which would result from the grinding action of the stirringblade against the walls of the reaction vessel. After completion of thereaction the resin was filtered onto a tared, coarse-fritted Btlchnerfunnel and washed successively with ethanol, water, methanol andmethylene chloride (using three portions of each solvent). The resin wassuction-dried and the amount of valine attached to the resin wasdetermined by amino acid analysis to be 0.02 mm./g. This is equivalentto a 50% utilization of the chloromethyl groups available in Bead B.This compares favorably with literature values of 20-40% utilization ofchloromethyl groups indicated for the chloromethylated polystyrene beadsknown in the prior art.

EXAMPLE 3 The procedure of Example 2 was repeated utilizing 120 mg.(0.377 mm.) of Boo-nitro argenine, 10.2 g. of Bead B, 30 ml. of ethanoland 0.0475 ml. of triethylamine for a reaction period of 71 hours andgave a utilization value of 31.6% (0.017 mm./g. of nitro argenineattached to the resin by amino acid analysis). This represents anextremely favorable utilization value since Boc-nitro argenine is knownto be a difiicult component in coupling reactions with solid-phasesupports.

EXAMPLE 4 Preparation of H-Leu-Ala-Gly-Val-OH via shaker flask I.H-Leu-Ala Gly-Val-resin ester.A total of 7.00 g. of Bead B containingBoo-valine as prepared in Example 2 was placed in a Merrifieldsolid-phase shaker vessel and treated according to the followingschedule:

. add 50 ml. of dioxane shake for 5 minutes filter solvent repeat steps1-3 two times add 50 ml. of 4N HCl-dioxane shake for 30 minutes filtersolvent add 50 ml. of dioxane shake for 5 minutes filter solvent add 50ml. chloroform shake for 5 minutes filter solvent repeat steps 1-3 twotimes add 50 ml. of methylene chloride shake for 5 minutes repeat steps1-3 two times 1. add 674 mg. (3.85 mm., 27.5 equiv.) of Boo-glycine in40 ml. CHgClg 2. shake 10 minutes ey le 1. add 793 mg. (3.85 mm., 27.5equiv.) of N,N'-dicyclohexylcarbodiimide dissolved in 10 ml. of CH CIadd 50 ml. of methylene chloride shake for 5 minutes repeat steps l-3two times add 50 ml. of absolute ethanol shake for 5 minutes repeatsteps l-3 two times The cycle was repeated using 728 mg. (3.85 mm.) ofBoc-alanine, and 890 mg. (3.85 mm.) of Boc-leucine. After the final stepin the Boc-leucine coupling cycle, the cycle was again repeated up tothe end of the second dioxane rinse step (step C).

II. H-Leu-Ala-Gly-Val-OH (cleavage reaction).-The air-dried resin wascombined with 70 ml. of trifiuoroacetic acid in a cleavage vesselconsisting of a 125 ml. dropping funnel with a fritted disc in a 29/42joint on the bottom, A stream of HBr was bubbled through the mixture forminutes. The liquid phase was collected in a round bottom flask and theresin was rinsed twice with 40 ml. of trifiuoroacetic acid, and twicewith a 50% mixture of trifiuoroacetic acid and methylene chloride. Thecombined filtrations were evaporated on a rotary evaporator to drynessat 35 C. The residue was dissolved in approximately 10 ml. of methylenechloride and the solution was again evaporated to dryness at 35 C. Thisprocedure was repeated two more times using 10 ml. of methylene chlorideeach time. The residue was then evaporated from water at 40 C. in thesame manner, using approximately 10 ml. of water each time. The residuewas then dissolved in approximately 10 ml. of water and treated with asmall amount of activated charcoal at approximately 3540 C. for 10minutes. The mixture was filtered directly into a 45 ml. lyophilizationbottle. On lyophilization, the clear, colorless solution produced 44.8mg. (yield 63%) of a white, powdery solid. Thin layer chromatography ofthe product showed one spot corresponding to that of authenticH-Leu-Ala-Gly-Val-OH synthesized with the Merrifield resin. Amino acidanalysis ratios were: Leu, 1.00; Ala, 1.00; Gly, 0.97; Val, 1.01.

11 EXAMPLE An experiment following the procedure of Example 4 wasperformed using Bead C. It was found that 7.00 g. of this resin produced28.3 mg. of material having one major spot and a trace of impurity inthe thin layer chromatography. The major spot corresponds with that ofauthentic H-Leu-Ala-Gly-Val-OH.

EXAMPLE 6 Preparation of H-Leu-Ala-Gly-Val-OH via column The column usedwas a chromatographic-like column consisting of a 50 ml. burette fittedwith a 14/20 joint at the top and a coarse, fritted disc at the bottom.Solvents were gravity-added to the column by means of a reservoir placedon top, and collected in a waste container at the bottom. During thecoupling reactions, Boo-amino acid-N,N-dicyclohexylcarbodiimide mixtureswere recirculater through the resin by means of a peristaltic pump andrecirculated through Compar (polyvinyl alcohol) tubing.

Experimental procedure Boc-valine loaded Bead B (2.5 g.) as prepared inExample 2 was placed in the column described above and treated accordingto the following schedule:

. rinse with 150 ml. of dioxane rinse with 150 ml. of 4 N HCl-dioxanerinse with 150 ml. of dioxane rinse with 150 ml, of chloroform rinsewith 150 ml. of triethylamine-chloroform rinse with 150 ml. ofchloroform rinse with 150 ml. of methylene chloride recirculate asolution of 35 mg. (0.2 mm., 4 equiv.) of Booglycine in 30 ml. methylenechloride for ten minutes add solution of 41.2 mg. (0.2 mm.) ofN,N-dicyclohexylcarbodiimide, and recirculate the mixture for two hoursi. rinse with 150 ml. of methylene chloride k. rinse with 150 ml. ofabsolute ethanol.

The cycle (steps a through k) was repeated using 37.8 mg. (0.2 mm.) ofBoo-alanine, and 46.2 mg. (0.2 mm.) of Boc-leucine'. After completion ofstep k in the Bocleucine coupling cycle, steps a through c were repeatedto remove the terminal Boc group on leucine.

Cleavage reaction The cleavage reaction and isolation of the finalproduct were carried out in exactly the same manner as described forExample 3, with the exception that 28 ml. of trifiuoroacetic acid wasused in this case. The reaction was also carried out in a comparativelysmaller vessel (-60 ml.).

The white solid obtained on lyophilization weighed 17.9 'mg. (71.1%yield computed as the acetate-dihydrate salt) and produced one majorspot and a very faint trace of impurity (slow) on thin layerchromatography. The major spot corresponds with that of authenticH-Leu-Ala-Gly- Val-OH. Amino acid analysis ratios were: Leu, 0.93; Ala,0.90; Gly, 1.00; Val, 1.06.

EXAMPLE 7 Preparation of L-leucyl-L-alanylglycyl-L-valine Attachment oft-BOC-L-value to resin.-A solution of t-BOC-L-valine (810 mg., 3.74mmol) in absolute alcohol (180 ml.) was added to 70 gm. of polystyrenecoated glass beads said polystyrene being cross-linked to 0.5% withdivinylbenzene and chloromethylated to a chlorine level of 0.056 mm./g.of coated glass bead which bead comprised about 99.22 weight percentglass and 0.78 weight percent polymer, (0.2% chlorine, 3.95 mmol) and0.47 ml. (3.36 mmol) of triethylamine. The mixture was refluxed gentlyfor 17 hours. After cooling to room temperature the beads were filteredusing a 150 ml. coarse fritted funnel and washed with ethanol, water,methanol and methylene chloride and dried under vacuum. The yield was 68gum. and amino acid analysis showed 22.7 ,umol of valine/ gm. of resincoated beads.

The detailed procedure for the synthesis of L-leucyl-L-alanyl-glycyl-L-valine in a column using 4.0 g. of the coated beadprepared above is described in the following schedule:

a. rinse with 3 ml. of dioxane;

b. rinse with 5 ml. of 4N HCl-dioxane (wait 1 minute before step c);

rinse with 3 ml. of dioxane;

rinse with 3 ml. of chloroform;

rinse with 3 ml. of 10% triethylamine-chloroform; rinse with 3 ml. ofchloroform;

rinse with 3 ml. of methylene chloride;

. treat alternatively with separate portions of- 1) 12 ml. of a solutioncontaining 2412 mm./l0 ml. of N,N' cyclohexylcarbodiimide in methylenechloride;

(2) 12 ml. of a solution containing 2.12 mm./10 ml. of t-BOC-L-glycinein methylene chloride (wait 8 minutes before step i);

i. rinse with 3 ml. of methylene chloride; j. rinse with 10 ml. ofmethanol.

The cycle (steps a through j) was repeated utilizing equivalentconcentrations of t- BOC-L-alanine and t-BOC- L-leucine respectively forthe t-BOC-L-glycine used above. A temperature of 38 C. was employed forthe above processes which temperature was maintained in the column byutilizing a water jacket around the column.

Cleavage of peptide from resin.To cleave the peptide from the resin, astream of HBr saturated with trifluoroacetic acid was passed through thecolumn for 2 hours. The excess HBr-trifluoroacetic acid was removed bynitrogen gas for 15 minutes. The peptide was eluted with 1N HCl,adjusted to pH 2.0 2.2. The purity of the product was shown to be98.61%. For the isolation of the tetrapeptide water was used for elutioninstead of 1N HCl and the yield was shown to be quantitative afterlyophilization. The product was shown to be identical to a known sampleof the above-titled compound.

EXAMPLE 8 Preparation of dinitro-bradykinin Synthesis oft-'BOC-nitro-arginyl-polymer.To a solution of t-BOC-L-nitro-arginine(450 mg., 1.41 mmol) in absolute alcohol ml.) was added 25 gm. of theEx. 7 resin coated glass beads (0.2% chlorine, 1.41 mmol) and 0.'-l8 ml.(1.27 mmol) of triethylamine. The mixture was refluxed gently for 40hours. After cooling, the beads were filtered and washed with ethanol,water, methanol and methylene chloride and dried in vacuum. Amino acidanalysis showed 11.0 mol of nitroarginine per gm. of resin coated head.

The detailed procedure for the synthesis of dinitrobradykinin in acolumn using 4.0 g. of the coated bead prepared above is described inthe following schedule:

a. rinse with 3 ml. of dioxane; b. rinse with 5 ml. of 4N HCl-dioxane(wait 9 minutes 'before step c);

. rinse with 3 ml. of dioxane; 1

. rinse with 5 ml. of chloroform;

. rinse with 5 ml. of 10% triethylamine-chloroform;

. rinse with 3 ml. of chloroform;

rinse with 3 ml. of methylene chloride;

. treat alternatively with separate portions of (1) 12 ml. of a soluitoncontaining 2.12 mm./ 10

ml. of N,N'-cyclohexane carbodiimide in methyl ene chloride;

13 (2) 12 ml. of a solution containing 2.12 mm./ 10 ml. oft-BOC-phenylalanine (wait 8 minutes before step i); i. rinse with 3 ml.of methylene chloride; j. rinse with 10 ml. of methanol.

The cycle (steps a through i) was repeated utilizing equivalentconcentrations of the following protected amino acids instead oft-BOC-L-phenylalanine in the order indicated:

t-BOC-L-proline, t-BOC-O-benzyl-L-serine, t-BOC-L-phenylalanine,t-BOC-L-glycine, t-BOC-L-proline, t-BOC-NO -L-arginine When employingthe last named amino acid, i.e., t-BOC- NO -L-arginine,dimethylformamide is substituted for methylene chloride in all instancesin the preparation. The foregoing procedure was conducted with thecolumn maintained at room temperature.

Cleavage of peptide from resin.To cleave the peptide from the resin, astream of HBr saturated with trifluoro acetic acid was passed throughthe column for 2 hrs. After removing the excess HBr-trifluoroacetic acidby nitrogen gas, the column was eluted with 50 ml. of water which waslyophilized to yield 34.4 mg. of dinitro-bradykinin, thin layerchromatography on silica gel plate using solvent system of nBuOI-I (1)EtOH (1), HOAc (1), H O 1) showed one major spot which has the same RFvalue as a known sample.

EXAMPLE 9 A suspension of grams of polystyrene coated glass beadscrosslinked to 0.5% with divinyl benzene said head containing 0.5 wt.percent resin and 95.5 wt. percent glass beads and are chloromethylatedto a chlorine level of 25.6 wt. percent chlorine, in 12 ml. of 80%dimethylformamide was mixed with a solution of 2 g. (41 mmole) of sodiumcyanide in 24 ml. of the same solvent. The suspension was sealed andstirred in a 118 C. oil bath for 20 hours. After being cooled toapproximately 60 C., the dark, tarry mixture was centrifuged, and thesediment washed with Water until the washings were colorless. The darkresidue was then washed with 1:3 dioxane-water (5 X 20 ml.), 1:1dioxane-water (5X 20 ml.), 3:1 dioxane-water (5 x 20 ml.), dioxane (3x20 ml.), ethanol (4x 20 ml.), and ether (4X 20 ml.).

After removal of the ether in vacuo, the dark material was suspended in15 ml. of a 1:1:1 mixture of 96% sulfuric acid, glacial acetic acid, anddistilled water. The mixture was heated in a 120 C. oil bath for hours,cooled to room temperature, and washed by decantation with water untilthe washings were neutral. The sediment was then successively washedwith: 1:3 dioxane-water (4X 20 ml.), 1:1 dioxane-water (4X 20 ml.), 3:1dioxane-water (4X 20 ml.), 1:3 dioxaneethanol (4X 20 ml.), 1:1dioxane-ethanol (3X 20 ml.), 1:3 dioxane-ethanol (3X 20 ml.), ethanol(4X 20 ml.), and ether (4X 20 ml.). After removal of the ether in vacuo,the tan residue weighed 1.3 grams.

This material was suspended in 5 ml. of toluene, 5 ml. of thionylchloride were added, and the suspension refluxed overnight withexclusion of moisture. After cooling to room temperature, the solventwas removed by decantation and thionyl chloride washed out with toluene.After several ether washes, the residue was made solventfree by dryingin vacuo. The ofi-white solid was suspended in 10 ml. of dry pyridineand 1.83 g. (7.55 mmole) of 2-deoxythymidine were added. The reactionvessel was sealed and stirred at room temperature for 22 hours.Methanol, 1 ml., was added, and the mixture 14 stirred for 22 hours atroom temperature. After removal of the solvent by decantation, the solidwas washed with pyridine (5 X 10 ml.), and ether (5x 10 ml.). Afterremoval of the ether in vacuo, the product weighed 1.29 g.

Reaction of 1.2 g. of this material with 0.165 (0.357 mmole) of3'-O-acetyl-2-deoxythymidine-S'-phosphate pyridinium salt and 155 mg.(0.71 mmole) of mesitylene sulfonic acid in 2 ml. of dry pyridine atroom temperature overnight produced, after washing (water, ethanol,ether) to remove excess reagents and non-bound prod ucts, 0.97 g. ofglistening white beads. Reaction of this product with doxane-aqueousamonia at room temperature overnight produced thymidine3'-5 thymidylate,3 mmol, as shown by high pressure anion exchange chromatography andultraviolet spectroscopy.

We claim:

1. A solid support medium comprising an essentially fluid impervious,shaped core material coated with a highly swellable polymer resinderived from styrene, phenol-S-trioxan, methyl methacrylate, vinylalcohol, cellulose, dextrin, vinyl toluene, vinyl xylene, vinyl naphthalene, vinyl ethyl benzene, a-methyl styrene, vinyl chlorobenzene, orvinyl dichlorobenzene, cross-linked in a range of from about 0.01 to2.00 percent with a crosslinking agent selected from the groupconsisting of divinyl benzenes, divinyl toluenes, divinyl xylenes,divinyl naphthalenes, and divinyl ethyl benzene, said polymer resincontaining reactive groups selected from the group consisting ofchloromethyl,

0 H noomomcmc-Q- (CHa)2S GHQ-Q, elem-@- c100 CHFQ i ii orcrnoQ, and c1-o2. The solid support of claim 1 wherein siad shaped core materialcomprises glass heads.

3. The solid support of claim 1 wherein said polymer coating comprisespolystyrene.

4. The solid support of claim 1 wherein said polymer resin is present inthe range of from about 0.2 to 20 weight percent and said core materialis present in the range of from about to 99.8 weight percent.

5. The solid support of claim 1 wherein said reactive groups comprisechloromethyl groups.

6. The solid support of claim 5 wherein said core material comprisesglass beads and said polymer resin comprises polystyrene cross-linked inthe range of from about 0.01 to 2.00% with di vinylbenzene.

15 16 7. The solid support of claim 1 wherein said shaped 2,876,1333/1959 Iler et a1. 117-400 S core material is spherical in shape.3,511,697 5/ 1970 Auken et a1. 117-46 FA References Cited WILLIAM D.MARTIN, Primary Examiner UNITED STATES PATENTS 5 D. C. KONOPACKI,Assistant Examiner 3,507,686 4/ 1970 Hagen'bach 117100S s c1, 3,252,9215/1966 Hansen et a1 26079.3

3,340,085 9/1967 Halasz et a1. 117 10o B 124 126 GB

